There is a need to recover certain cell types or certain molecular structures from mixed populations of cells or molecular structures.
Immunoselection is a generic term which encompasses a variety of techniques for the separation of cells or molecular structures bearing function related or lineage specific antigenic determinants. The specificity of the selection process is conferred by antibody or antibody-like molecules, haptens or lectins which interact with the specific cell surface or molecular structure antigenic determinants.
In recent years the biotin-avidin system has been applied to a variety of analytical and preparative procedures and has found wide spread use in immunoselection techniques for cell separation.
In general, two types of immunoselection techniques are available. "Negative selection" involves the removal of a specific subpopulation of cells from a heterogenous mixture of cell types. Using this method of separation, one can obtain an enriched but not pure preparation of remaining cells since all cell types in the original mixture that are negative for the selection antigen will be recovered. The second method, "positive selection" involves the specific targeting and recovery of cells expressing the desired specificity from a heterogenous population of contaminating cells. Positive selection techniques can provide a highly enriched or even pure population of the desired antigen-positive cells, in contrast to negative selection methods in which all cell types that are target antigen-negative will be recovered.
Biotinylated antibodies against rat thymocytes have been used to remove such thymocytes from a cell mixture by interaction with avidin covalently coupled to nylon meshes but removal of the thymocyte cells from the avidin was not attempted (Jasiewicz et al., Exp. Cell Res. 100:213-19, 1976).
T cells have been removed from spleen cell preparations by reaction with biotinylated monoclonal antibody directed against T cell antigen followed by panning on avidin, coated plates but the T cells were not removed from the plates (Prud'homme et al., J. Exp. Med. 159:463-78, 1984). U.S. Pat. No. 4,298,685 also describes a negative selection process using biotin-avidin.
Biotinylated monoclonal antibodies have been used in combination with avidin coated sheep erythrocytes to form rosettes of selected cells which are then separated on density gradients (Wormmeester et al., J. Immunol. Methods 67:389-94, 1984). This procedure reportedly allows for both positive and negative selection of cell populations. However, positively-selected cells are coated with sheep erythrocytes which could affect their activity.
All of the described methods rely upon the direct interaction of biotin with avidin or streptavidin bound to a solid support matrix during the immunoselection process to achieve cell separation, whether by a process of positive or negative selection. However, because of the very high affinity of the biotin-avidin interaction, the process is essentially irreversible and it is difficult to recover intact, functional cell populations away from the solid support matrix.
Various attempts have been made to circumvent this difficulty including the use of biotin analogues with lower affinity for avidin (Basch et al., J. Immunol. Methods 56:269-80, 1983) or the use of avidin-Sepharose matrices with reduced affinity for biotin (U.S. Pat. Nos. 4,253,996 and 4,276,206). These methods reportedly yield an operable avidin/biotin based PG,6 positive selection procedure. However, the efficiency of isolating the desired components is variable.
Additional antibody layers on targeted cells have also been used such that the linkage to the cell can be mechanically disrupted at the lower affinity antigen-antibody interaction site (Berenson et al., J. Immunol. Methods 91:11-19, 1986; U.S. Pat. No. 5,215,927; International Patent No. WO 87/04628). This process, however, does not allow for the specific elution of the selected, retained cell population since any contaminating cells non-specifically adsorbed to or mechanically trapped by the avidin-derivatized solid phase may also be released by the mechanical agitation elution process. Further the mechanical agitation process may damage the cells.
International Patent Application Publication No. WO 92/16841 discloses a non-immune, reversible binding displacement system for the detection of compounds in a solution. Specifically, the application discloses the attachment of a releasable ligand, a binding partner for the releasable ligand, an analyte of interest, an analytically detectable (reporter) group and at least one binding partner for the analyte to an insoluble phase. A displacer ligand is added to the solution which displaces the releasable ligand along with some portion of the reporter-labeled complex which may or may not contain the analyte of interest so as to detect the presence of the analyte. This method discloses the use of biotin analogues with lower affinity for avidin as the releasable ligand.
Both positive and negative immunoselection methods for cell or molecular structure separation would be improved by a procedure which allowed for a high degree of specificity and selectivity in targeting of cells and provided for an efficient means of recovering those targeted cells from the solid support under conditions which minimized damage to the cellular structure and/or molecular integrity.